It is well known to those familiar with the field that in recent years several continuous cell culture processes, also called continuous perfusion processes, have been established with great commercial success. However, the isolation process following continuous perfusion fermentation is generally a batch process, and is physically and logistically separated from the continuous upstream process. In these processes, the main purpose of the isolation step is to capture the product out of high volumes of relatively dilute culture supernatant. Concentration of the product has to be emphasized with respect to process logistics and space requirements, while simultaneous removal of contaminants (purification) is important to minimize the required number of further downstream purification steps.
FIG. 1 shows a schematic of a typical state-of-the-art isolation process from continuous perfusion fermentation, as it is well known to those familiar with the field. The continuous perfusion fermentation system comprises a cell retention device (1), which keeps most of the cells producing the product in the fermentation system. A continuous harvest stream from the continuous perfusion system, still containing some cells, debris and other particles is pumped using a harvest pump (2) into large collection vessels (3), such as stainless steel tanks. These harvest storage vessels usually have to be cooled in order to keep product losses due to degradation within a feasible range.
Once a specified volume has been collected, which is typically after 1-4 days or more, the harvest collection vessels are disconnected from the sterile fermentation vessel and the collected material is designated as one harvest batch. The next step is to remove cells, debris and particles (step 2). In industrial scale this is typically done using centrifugation (4) followed by dead-end membrane filtration (5), or by dead-end depth-filtration (6) followed by dead-end membrane filtration (7). Another technique sometimes used is tangential flow (or “crossflow”) microfiltration. In any case, the product of the particle removal process is a batch of clarified tissue culture fluid, or cTCF (8). More details on particle separation for biotechnological products can be found in standard textbooks, such as Biotechnology, Vol. 3, Bioprocessing, Wiley-VCH, 2 edition (1996), ISBN: 3527283137.
In the next step (step 3), the batch of clarified tissue culture fluid is further processed to concentrate and, if possible, purify the product. This is typically done by crossflow ultrafiltration or by packed bed chromatography.
In the case of crossflow ultrafiltration, the cTCF is pumped into the recycle tank (9) of the system. A pump is used (10) to push the material through a crossflow ultrafilter. The product is retained by the membrane and recycled as retentate into the recycle tank, while water and smaller contaminants are pushed through the membrane into the permeate (11) due to the trans-membrane pressure generated by the pressure drop in the ultrafiltration module. In each passage through the filter, the cTCF therefore gets more concentrated, and the total cTCF volume is reduced until a desired concentration factor is reached. Once the desired concentration factor is reached, the process is stopped, and the remaining concentrate volume (isolate) is drained from the system and collected. More details on crossflow ultrafiltration for concentration of biotechnological products can be found in standard textbooks, such as Biotechnology, Vol. 3, Bioprocessing, Wiley-VCH, 2 edition (1996), ISBN: 3527283137.
In case of packed bed chromatography, the cTCF is pumped over a chromatography column (12) containing a packed resin bed. The product binds to the resin and is then eluted in usually concentrated and purified form (isolate, 13) using a suitable elution buffer (14), after which the column is cleaned and regenerated using adequate buffers and cleaning solutions (14).
Other chromatography variants that have been proposed for concentration/purification of cTCF are expanded bed chromatography and membrane chromatography. Expanded bed chromatography can process particle containing solutions. However, filtration of the isolate after chromatography is still required, although filtration areas are reduced. Membrane chromatography utilizes stacks of modified microfiltration membranes instead of packed resin beds. The advantage is that mass transfer is largely convective rather then diffusive, which allows faster separation. Otherwise, the process is typically equivalent to standard packed bed chromatography. More details on chromatography for concentration and purification of biotechnological products can be found in standard textbooks, such as Protein Purification, Principles, High-Resolution Methods, and Applications, Wiley-VCH, 2. Edition (1998), ISBN 0-471-18626-0.
Often, the bulk isolate is then frozen and stored for later use in further downstream purification steps.
Thus, as described above, the isolation process is generally a batch process, and is physically and logistically separated from the continuous upstream process. Also, while fermentation has to be performed sterile, isolation (i.e. particle removal and concentration/purification) is essentially performed clean, but not sterile.
The state-of-the-art processes as described above have a number of problems:
P1. Yield losses and potential quality reduction due to high product residence time. The harvest from the continuous perfusion fermentation needs to be collected and stored over significant periods of time, as outlined above, before an isolation batch can be processed. Collected harvest, although chilled, still provides a detrimental milieu for complex and inherently unstable protein products. Therefore, significant product losses occur, which reduces plant capacity and increases cost of goods. Furthermore, the product quality can be adversely affected.
P2. Large cold room facilities or cooled vessels are required for intermediate storage of large harvest volumes, leading to high capital costs and negating the cited compactness and mobility advantage of perfusion fermenters.
P3. Conventional concentration/purification technologies (e.g., ultrafiltration, packed bed chromatography) have relatively low volumetric throughput, significant turnaround times and are relatively labor intensive. As a result, typically not more then 1 batch process is performed per day.
P4. Moreover, current isolation processes and methods have logistical difficulties dealing with the varying process volumes in fermentation plants involving more then one fermenter. In large-scale continuous perfusion plants, a varying number of fermenters are operational.
P5. Furthermore, state-of-the-art isolation processes are operated clean, but can not be operated sterile. This often leads to a significant number of rejected batches due to microbial load issues.
P6. Utilization of disposables, such as disposable filters, assemblies, bags, etc., although very desirable in the production of human parenterals (e.g., to avoid cleaning and cleaning validation and other issues) is very costly, and in fact often not economical.
Accordingly, it is an object of the present invention to provide a integrated, continuous protein separation process that is capable of operating for sustained periods of time under sterile conditions.